Abstract
Embryonic stem (ES) cell self-renewal and pluripotency are maintained by several signaling cascades and by expression of intrinsic factors, such as Oct3/4 and Nanog. The signaling cascades are activated by extrinsic factors, such as leukemia inhibitory factor, bone morphogenic protein, and Wnt. However, the mechanism that regulates extrinsic signaling in ES cells is unknown. Heparan sulfate (HS) chains are ubiquitously present as the cell surface proteoglycans and are known to play crucial roles in regulating several signaling pathways. Here we investigated whether HS chains on ES cells are involved in regulating signaling pathways that are important for the maintenance of ES cells. RNA interference-mediated knockdown of HS chain elongation inhibited mouse ES cell self-renewal and induced spontaneous differentiation of the cells into extraembryonic endoderm. Furthermore, autocrine/paracrine Wnt/beta-catenin signaling through HS chains was found to be required for the regulation of Nanog expression. We propose that HS chains are important for the extrinsic signaling required for mouse ES cell self-renewal and pluripotency.
Highlights
Research for Evolutional Science and Technology of Japan Science and Technology Agency, Kawaguchi Center Building, 4-1-8, Hon-cho, Kawaguchi, Saitama 332-0012, Japan
We have demonstrated that EXT1-deficient mouse ES (mES) cells, which express greatly reduced levels of Heparan sulfate (HS) chains, proliferate slowly and differentiate spontaneously into the extraembryonic endoderm lineage
We demonstrated that autocrine/paracrine Wnt/-catenin signaling through HS chains was necessary for maintenance of Nanog expression, self-renewal, and pluripotency in mES cells even in the presence of leukemia inhibitory factor (LIF)/ STAT3 and bone morphogenic proteins (BMPs)/Smad signaling
Summary
Cell Culture and Transfection—R1 [27] and E14TG2a [28] mES cell lines were maintained on mouse embryonic fibroblasts (MEFs) inactivated with 10 g/ml mitomycin C (Sigma) in ES medium (Dulbecco’s modified Eagle’s medium supplemented with 15% FBS {Hyclone}, 1% penicillin/streptomycin {Invitrogen}, 0.1 mM mercaptoethanol {Invitrogen}, and 0.1 mM nonessential amino acids {Invitrogen}) with 1000 units/ml LIF (Chemicon). The mES cells were harvested, replated at 1 ϫ 106 cells on gelatin-coated feeder-free 60 mm tissue culture dishes (Iwaki) in ES medium with LIF, and incubated for 16 h. Molecular Size Analysis of HS Chains—One day after transfection, mES cells were harvested, replated at 1.5 ϫ 106 cells per well in 6-well 0.2% gelatin-coated plates and incubated in sulfate-free ES medium with LIF, puromycin and 100 Ci/ml Na235SO4 (ARC). Self-renewal Assay—Two days after transfection, mES cells were harvested and replated at 1 ϫ 104 cells per gelatin coated 60 mm tissue culture dish in ES medium with LIF. The relative amounts of each mRNA were normalized by -actin mRNA in the same cDNA
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